Kinetic methods of analysis are known for measuring concentrations of an analyte in a sample. Kinetic analyses are based on the principle that a reaction between the analyte to be measured and a suitable reagent need not be completed in order to determine the initial concentration of the analyte. Instead, the rate at which the reaction occurs, that is, at which a reaction product is formed, is measured at a preselected time after the start of the reaction. The rate of the reaction so determined is proportional to the initial concentration of the analyte and such concentration may be determined from a calibration curve which relates reaction rate to initial analyte concentration.
Two approaches to kinetic analysis are generally well known in the art. In each approach, a reaction product signal is developed which is proportional to the quantity of reaction product formed by an analyte and reagent in a sample. As the reaction progresses, the reaction product signal varies in accordance with the quantity of reaction product formed.
In a first type of kinetic analysis, sometimes referred to as two point or multipoint rate analysis, the reaction product signal is measured at two or more predetermined times or "points" after the beginning of the reaction. The reaction product signals measured over the time period covered by the predetermined times is analyzed to determine the slope of a line which best fits the measured reaction product signals. The slope is indicative of an average reaction rate during such time period. The average reaction rate so measured may then be compared to a calibration curve to determine the initial analyte concentration in the sample. Examples of apparatus employing two point or multipoint kinetic analysis techniques include the ABA-200.RTM. biochromatic analyzer from Abbott Laboratories, the Centrifichem.RTM. System 400 from Union Carbide Corporation and the Cobas.RTM. Bio from Roche Analytical Instruments.
A second approach to kinetic analysis, sometimes referred to as derivative analysis, may be performed electronically by differentiating the reaction product signal to form a rate signal which is proportional to the rate of change of the quantity of reaction product formed during the reaction. The rate signal is measured at a predetermined time after the start of a reaction and may be compared to a calibration curve to relate the differentiated reaction rate signal to an initial analyte concentration. The ASTRA.TM. Automated Stat/Routine Analyzer System manufactured by Beckman Instruments, Inc. includes modules which perform kinetic analysis using such a derivative technique.
Kinetic analysis methods have been widely used for in vitro quantitation of creatinine to estimate total renal function. A reaction known as the Jaffe reaction has been the method of choice for such analysis and refers to a reaction of creatinine with picric acid in an alkaline medium to form a red colored creatinine-picric acid complex. Using the Jaffe reaction, it is possible to assess the function of the kidney with a creatinine clearance test that measures the relative amount of creatinine excreted in the urine with respect to the serum creatinine concentration. The creatinine clearance test provides a more sensitive indication of renal function than quantitation of serum or urine creatinine alone.
Unfortunately, the Jaffe reaction is not specific to creatinine, that is, the Jaffe reaction performed in the presence of one or more interfering substances or "interferents" may result in a positive or negative bias in the Jaffe reaction product. Although a number of substances have been identified which produce a bias in the Jaffe reaction result, it has been recognized for some time that acetoacetate is the most frequent analytical interferent in creatinine kinetic assays where the reaction rate is measured at a time when interferents having reaction rates generally faster than creatinine are still contributing significantly to the reaction product. Acetoacetate interference produces a positive bias in the Jaffe reaction product. Various disturbances in normal metabolism may produce levels of acetoacetate in patient serum which may substantially effect the accuracy of a creatinine clearance test. In view of the importance of determining creatinine concentration in estimating total renal function, it is clear that creatinine levels should be measured so as to minimize or eliminate acetoacetate interference.
Prior kinetic analytical methods have attempted to minimize acetoacetate interference in creatinine assays by delaying the measurement of the reaction rate to a time at which the contribution of acetoacetate to the formation of the reaction product is small as compared to the amount of reaction product formed by creatinine. Such a method is disclosed in U.S. Pat. No. 3,682,586 to Ertingshausen et al. for "Process for the Determination of Creatinine Body Fluids". However, such methods merely reduce acetoacetate interference in the creatinine determination and do not indicate that acetoacetate bias may be present in the measured reaction rate. The prior methods also do not compensate or correct the measured reaction rate and resulting creatinine concentration for acetoacetate interference. Furthermore, the delay required before reading the reaction rate reduces the number of assays which may be performed and thus the throughput of the kinetic assay apparatus. Moreover, delaying the measurement of the reaction rate requires that the reaction rate be measured after a significant portion of creatinine has already reacted with the Jaffe reaction reagent, decreasing the sensitivity and/or accuracy of the kinetic assay.
Thus, there is a need for a method and apparatus for the kinetic assay of creatinine which indicates that the result of the assay is influenced by the presence of acetoacetate. There is also a need for a kinetic assay method and apparatus which corrects for the influence of acetoacetate in determining creatinine concentration. There is a further need to provide a kinetic assay method and apparatus which relatively rapidly performs creatinine kinetic assays while correcting for the influence of acetoacetate in creatinine concentration determinations. Furthermore, there is a need for a creatinine kinetic assay method and apparatus which measures reaction rate while a significant concentration of creatinine remains in the sample undergoing analysis to thereby improve the sensitivity and/or accuracy of the creatinine concentration determination.